The seventh group was daily treated with 1 mg Cands /kg/day  and was intraperitoneally injected with 20 mg 3-NP/ kg, from the 3rd till the 9th day. The present results revealed that 3-NP injection induced a considerable body weight loss, impaired memory and locomotor activity, reduced striatal monoamine levels. Furthermore, 3-NP administration remarkably increased striatal malondialdehyde and nitric oxide levels, whereas markedly decreased the total antioxidant capacity. Moreover, 3-NP significantly upregulated the activities of inducible nitric oxide synthase and caspase-3 as well as the Fas ligand, in striatum. On the contrary, Safr and Cands remarkably alleviated the above-mentioned 3-NP-induced alterations. In conclusion, Safr and Cands may prevent or delay the progression of HD and its associated impairments through their antioxidant, anti-inflammatory, anti-apoptotic and neuromodulator effects.3-nitropropionic acid (3-NP) is a potent mitochondrial inhibitor mycotoxin. Systemic administration of 3-NP can induce Huntington’s disease (HD)-like symptoms in experimental animals. Safranal (Safr) that is found in saffron essential oil has antioxidant, anti-inflammatory and anti-apoptotic actions. Candesartan (Cands) is an angiotensin receptor blocker that has the potential to prevent cognitive deficits. The present study aims to investigate the potential neuroprotective efficacy of Safr or Cands in 3-NP-induced rat model of HD. The experiments continued for nine consecutive days. Rats were randomly assigned into seven groups. The first group (Safr-control) was daily intraperitoneally injected with paraffin oil. The second group (Cands- and 3-NP-control) daily received an oral dose of 0.5% carboxymethylcellulose followed by an intraperitoneal injection of 0.9% saline. The third and fourth groups received a single daily dose of 50 mg/kg Safr  and 1 mg/kg Cands , respectively. The sixth group was daily treated with 50 mg Safr kg/day  and was intraperitoneally injected with 20 mg 3-NP/ kg, from the 3 Pathologically, HD is caused by an expansion of cytosine–adenine–guanine (CAG) trinucleotide repeats in huntingtin gene exon causing an abnormal translation of its protein ,4. The mutant huntingtin protein causes disruption of the striatum initially followed by the cortex and the hippocampus . Several mechanisms were suggested to explain the pathogenesis of HD such as oxidative stress, mitochondrial or synaptic dysfunction, defects in energy metabolism and apoptosis of striatal neurons, but the exact mechanism is not clear so far .Huntington’s disease (HD) is a fatal neurodegenerative disease characterized by significant motor and cognitive impairments. It is associated with psychiatric and behavioral disturbances. Unfortunately, no cure has been recognized yet to slow down or reverse its progression . It is a potent mitochondrial inhibitor mycotoxin. Systemic administration of 3-NP to experimental animals produces HD-like symptoms via developing bilateral striatal-specific lesions. 3-NP is a non-competitive inhibitor of mitochondrial succinate dehydrogenase , interfering with the electron transport cascade and oxidative phosphorylation, resulting in cellular energy deficit and oxidative stress .3-Nitropropionic acid (3-NP) is a natural toxin obtained from various plants and fungi Crocus sativus L., Iridaceae, one of the most expensive herbs used as a natural additive in cooking to enhance flavor, color and aroma. It is used also in the traditional medicine for its hypolipidemic, anti-cancer and antidepressant properties . Safranal (Safr) is the main component of saffron essential oil that causes its distinctive aroma. It has been shown that Safr has several pharmacological effects such as antioxidant, anti-inflammatory, gastro-protective and anti-apoptotic effects . Furthermore, Safr could improve cognitive and memory deficits and exerts numerous neuropharmacological properties as anxiolytic, anticonvulsant and antidepressant . Several studies have reported that Safr may be an effective treatment for various neurodegenerative diseases such as Parkinson’s disease  and Alzheimer’s disease .The use of medicinal plants rich in antioxidant phytochemicals as potential protective agents against several brain diseases has received growing attention. Their protective mechanism is via scavenging reactive oxygen species (ROS) and detoxifying potent genotoxic oxidants. Saffron is the dry red stigma of . Angiotensin II, the main effector in RAS, contributes mainly to the impairment of neurovascular coupling impairments . Ang II type 1 receptor (AT1R) blockers (ARBs) have been reported to be useful in diminishing cognitive deficits linked to Post-Stroke Cognitive Impairment (PSCI), Alzheimer’s Disease, Parkinson’s Disease, and Vascular Cognitive Impairment (VCI) . A recent study reported that dysregulated brain RAS may be implicated in neurodegeneration due to neuroinflammation, oxidative stress and aging-related pathophysiological changes .The renin-angiotensin system (RAS) is one of the most important systems in the body that regulates blood pressure and fluid homeostasis . Candesartan (Cands), the strongest AT1R antagonist can cross the blood-brain barrier to prevent traumatic brain injury and ischemia . Cands represents a promising possibility for the treatment and prevention of age-related memory impairment .ARBs can also ameliorate irradiation-induced brain stroke via protecting the cerebrovascular flow and reducing the risk of cognitive impairment . Thus, the present study aimed at investigating the possible protective effect of Safr and Cands against 3-NP induced mitochondrial, biochemical, behavioral and histological changes in the striatum of male Wistar rats.In the light of the above-mentioned beneficial effects, we hypothesize that the use of Safr or Cands may possibly ameliorate 3-NP-induced striatal damage in rats. To our knowledge, no study was designed to study the potential neuroprotective effect of Cands against 3-NP induced HD symptoms. However, only one recent study aimed to only investigate the effect of Safr on the 3-NP induced behavioral and oxidative stress parameters in HD rat model Pure Safranal (Safr), Cands (Candesartan), 3-NP (3-nitropropionic acid), paraffin oil, 0.5% carboxy methyl cellulose (CMC), thiobarbituric acid (TBA), vanadium chloride, sulfanilamide, N-(1-naphthyl) ethylenediamine, Folin-Ciocalteau reagent and bovine serum albumin (BSA) were obtained from Sigma . All other chemicals were of the highest analytical grades commercially available.To prepare a dose of 3-NP (20 mg/kg/day), each 20 mg of 3-NP was dissolved in 2.0 mL of 0.9% saline.A 50 mg of Safr was dissolved in 2.0 mL of paraffin oil to prepare the dose of Safr 50 mg/kg/day.Finally, 1 mg of Cands was dissolved in 2.0 mL of 0.5% CMC to prepare the dose of Cands 1 mg/kg/day.For all the applied drugs, the volume of administration was adjusted to 2.0 mL/kg body weight.3), with 4 rats per cage. The rats were kept under controlled conditions of constant temperature (23 ± 2°C), humidity (55 ± 5%) and 12 h/12h light/dark cycle (light on from 6:00 am to 5:59 pm and light off from 6:00 pm to 5:59 am), with ad libitum access to standard rodent chow and water. After one week of acclimatization, 280 rats were divided into seven main groups with 40 rats per group, as follows  injected with paraffin oil (PO), daily for 9 days, as negative control group for Safr group.rd day to the 9th day, as negative control group for Cands and 3-NP groups.The second group was pretreated orally with 0.5% CMC, daily for 9 days and was intraperitoneally injected with saline daily from the 3.The third group (Safr-treated) received intraperitoneal injection of Safr 50 mg/kg/day for 9 days .The fourth group (Cands-treated) was orally administered with Cands 1 mg/kg/day for 9 days rd till the 9th day .The fifth group (3-NP) received IP injection of 3-NP 20 mg/kg/day from the 3st till 9th day, whereas intraperitoneally injected with 3-NP 20 mg/kg/day from the 3rd day to the 9th day.The rats of the sixth group (Safr+3-NP) were intraperitoneally injected with Safr 50 mg/kg/day from the 1st till 9th day whereas intraperitoneally injected with 3-NP 20 mg/kg/day from the 3rd day to the 9th day.The rats of the seventh group (Cands+3-NP) were orally administered with Cands 1 mg/kg/day from the 1At the end of the experimental period, behavioral tests  were executed. In addition, the total body weight was recorded. Rats were sacrificed by decapitation and the striata were separated from the isolated brains. Tissue samples were stored at −80°C for further analysis.The current investigation complies with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health  and was approved by the Ethical Committee for Animal Experimentation at the Faculty of Pharmacy, Cairo University, approval no.: BC (1235).Open field test (OFT): The test was carried out to assess possible effects on locomotor activity during the morning daylight in a quiet lab, to avoid interference with any external stimuli. The OFT was executed in a square wooden arena (100 × 100 × 25 cm3). The clean floor was divided by black lines into 25 small squares (20 × 20 cm2) . The test started on the day 10 after treatment, at 8.00 am. Rats were placed individually into the central point of the open field and observed during five minutes test session for latency period "the time interval in seconds (s) between placing the animal at the middle of the arena until the decision of the animal to move", ambulation frequency "the number of squares crossed by the animal per minute", grooming frequency "the number of face washing and scratching with the hind leg and licking of the fur and genitals per 5 min" and rearing frequency "the number of times the animal stood stretched on the hind limbs with or without fore limbs support per 5 min". After each session, the floor of the OF was cleaned accurately from the urine spots and fees with 70% alcohol.Elevated plus maze test: The test was used to examine anxiety and spontaneous motor activity. The stainless-steel apparatus consists of 4 crossed arms that painted with matte black. Two open arms (50×10×30 cm3) and two closed arms (50×10×30 cm3) and the maze was elevated 65 cm above the floor. The test started on the day 10, after treatment, at 1:00 pm. The rat was placed in the center of the maze and the number of entries in open and closed arms, as well as the time the animal spent in the open and closed arms during a period of 5 minutes test session were recorded. The degree of avoidance of the open arms of the maze has been considered as a measure of strength of fear drive ,25.Passive avoidance test: to evaluate learning and spatial memory, an apparatus consisted of two equal-sized compartments (25 × 25 × 25 cm3), including a light and a dark compartment with a grid floor and Plexiglas walls that were separated by a guillotine door was used. To accustom rats to the apparatus, all rats were placed into the shuttle box for 1 day before main experiment for five min. On the 9th day of treatment, training session started at 6:00 pm. In the training session, each rat was individually put into the light compartment for 60s. After opening the guillotine door and the entrance of the rat into the dark compartment, the door was closed and a 0.5 mA with 50 Hz foot electric shock was delivered for 2s, through the grid floor. After 20s, the rats were transferred to their cage. On the 10th day of treatment, at 6:00 pm, the test session was done. In the test session, rats were placed in the light compartment. The step-through latency to enter the dark compartment was measured as a positive index of memory function. The delay in entering the dark compartment was recorded to a maximum of 3 minutes. The latency to enter the darkened chamber was measured for both acquisition (time from first exposure to the lighted chamber until the rat entered the darkened chamber) and retention (time from entry to lighted chamber until the rat entered the darkened chamber 24 hours later) .Each experimental group was divided into 5 subsets; the first subset (n = 10) was used to assess striatal caspase-3 (Casp-3), Fas ligand (Fas-L) and inducible nitric oxide synthase (iNOS) protein levels by Western blot technique. The second subset (n = 6) was used to measure the gene expression level of monoamine oxidases (MAO-A) and (MAO-B) via quantitative reverse transcription polymerase chain reaction (qRT-PCR) technique. The third subset (n = 10) was used for estimating oxidative stress parameters, monoamine levels and activities of MAO and acetylcholinesterase (AChE). The fourth subset (n = 10) was used for the analysis of mitochondrial complexes and comet assay. The last subset (n = 4) was used for striatal histopathological examination.. Nitric oxide (NO) level was determined by Griess method . Total antioxidant capacity (TAC) was measured using a commercial colorimetric kit  according to the manufacturer’s instructions.Striatum was weighed and 10% (w/v) homogenate was prepared in ice-cold 1.15% potassium chloride (KCl) for the determination of oxidative stress parameters. Malondialdehyde (MDA) content was determined as a biomarker of lipid peroxidation, using thiobarbituric acid (TBA) as described by Uchiyama and Mihara , MAO activity  and monoamine levels  , using the fluorometric methods.In addition, striatal tissue was homogenized in cold acidified n-butanol for analyzing AChE activity Total ribonucleic acid (RNA) was isolated from brain striatum using Qiagen tissue extraction kit (USA) according to the manufacturer’s instructions. The concentration and quality of the obtained RNA were assessed spectrophotometrically through measurement of the A260/A280 ratio using NanoDrop2000 . The total RNA was used for complementary DNA (cDNA) conversion using high-capacity cDNA reverse transcription kit  according to the manufacturer’s instructions. Quantitative real time PCR (qPCR) was performed using SYBR Green PCR Master Mix  with software version 3.1 . The qPCR assay with the primer sets were optimized at the annealing temperature. The sequences of the primers were as follows:.The thermal cycle protocol consisted of initial denaturation at 95°C for 10 min followed by 40 cycles with 15s denaturation at 95°C and 1 min annealing at 60°C /extension at 72°C. The changes in target gene expression were calculated using the comparative threshold cycle (Ct) method and presented as fold change The western blot method was done using V3 Western Workflow™ Complete System, Bio-Rad® Hercules, California, USA). Briefly, 5 mg of striatum tissue was homogenized in Radioimmunoprecipitation assay (RIPA) buffer, then centrifuged at 12,000 rpm for 20 min. The protein concentration for each cell lysate was determined using Bradford assay. Equal amounts of protein  were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked in Tris-buffered saline (TBS) buffer, 3% Bovine serum albumin (BSA) and 0.1% Tween 20 at room temperature for 1 h and incubated with Casp-3, Fas-L and iNOS primary antibodies, supplied by Thermoscientific , overnight at pH 7.6 at 4°C with gentle shaking. After washing, peroxidase-labeled secondary antibodies were added, and the membranes were incubated at room temperature for 1h. Image analysis software was used to read the band intensity of the target proteins against control sample after normalization by β-actin on the Chemi Doc MP imager.. The kit uses decylubiquinone, an analog of ubiquinone, as an electron acceptor that gets converted to decylubiquinol through the catalytic activity of complex I. The complex I dye absorbs light at 600 nm in its oxidized form and accepts electrons from decylubiquinol. Complex I activity was determined colorimetrically by recording the change in absorbance of reduced complex I dye at 600 nm. Complex I activity was calculated from the equation:2 –t1 (min), p is the mitochondrial protein (μg) and D is the dilution factor.Complex I (NADH:ubiquinone oxidoreductase) activity was measured using mitochondrial Complex I Activity Colorimetric Assay Kit , according to Ansari et al. Then the net complex I activity in the sample was calculated by subtracting the activity in reaction without rotenone minus the activity in reaction with rotenone. One unit of complex I is the amount of enzyme that causes the reduction of 1 μmol of the dye per min at pH 7.4 at room temperature.. Each well in the kit has been coated with an anti-complex II monoclonal antibody which purifies the enzyme from a complex sample. After purification, the production of ubiquinol by the enzyme results in the reduction of the dye 2,6-diclorophenolindophenol with a decrease in its absorbance at 600 nm. The activity of complex II (mOD/min) is the mean of measurements obtained with enzyme minus the rate obtained without enzyme.Complex II (succinate dehydrogenase) activity was measured by complex II enzyme activity microplate assay kit , as described by Horowitz et al. , using a kit purchased from Bio-vision, Milpitas, CA, USA. This method is based on monitoring the decrease in absorbance at 550 nm of ferrocytochrome-c caused by its oxidation to ferricytochrome-c where its activity was calculated in nmol cytochrome-c oxidized/min/mg protein.Complex IV (cytochrome-c oxidase) activity was assayed as described by Storrie and Amadden .Briefly, fully frosted slides were pre-coated on each end with 100 μl of 0.8% agarose in phosphate-buffered saline (PBS) and left at room temperature for 20 minutes. About 10,000 cells were mixed with 70 μl of 1% low-melting-point agarose in PBS. The mixture was immediately spread onto each end of a pre-coated slide and covered with a fresh glass coverslip. After lysis, denaturation, electrophoresis, neutralization and staining, the slides were examined with a fluorescence microscope. For each slide, 100 cells were counted at least twice. The comets were captured with an Olympus fluorescent microscope equipped with a charge-coupled device (CCD) camera, and the images were quantitatively evaluated for the percentage of DNA damage in the tail, tail length (TL) and tail moment (TM) using CASP software Striatum samples were kept in 10% formol saline for 24 h. Samples were washed with saline and dehydrated in serial dilutions of alcohol. Specimens were cleared in xylene and embedded in paraffin at 56°C for 24 h. Paraffin wax tissue blocks were prepared for sectioning at a thickness of 4 μm. The sections were deparaffinized and stained by hematoxylin and eosin (H & E)  for examination by the light microscope.Quantitative data were expressed as mean ± standard error of mean (SEM). One way analysis of variance (ANOVA) was used for comparing different groups. Duncan’s test was applied to study statistical differences among the experimental groups. All analyses were performed using Statistical Package for the Social Sciences (SPSS) version 18 for Windows  and differences were considered statistically significant at p<0.05 for all tests.In general, 3-NP injection caused marked reduction in the total body weight, the levels of monoamines and mitochondrial complexes were accompanied with remarkable elevations in the levels of monoamine oxidases, nitric oxide, caspase-3 and lipid peroxidation in the striatum of rats. Additionally, notable instabilities in the studied behavioural parameters and histopathological alterations were observed in the 3-NP treated rats.P = 0.000) reduced by about 20%. Though, as compared to the 3-NP group, the body weight showed remarkable elevations in rats of Safr+3-NP (P = 0.004) and Cands+3-NP (P = 0.000) groups by 11% and 13%, respectively.The changes in body weight of all groups are displayed in P = 0.000) in the latency period to step through the dark area by 87% and 91%, respectively, as compared to the CMC control group. In contrast, after 24h and 48h, rats of Safr+3-NP and Cands+3-NP groups showed significant elevations (P = 0.000) in the step-through latency after 24h and 48h by (6.8 & 6.3 folds) and (10.2 & 9.4 folds), respectively, as compared to the rats of 3-NP group.The data of PA test parameters after 24 h and 48 h are presented . After oIn On the other hand, Safr+3-NP- and Cands+3-NP-treated groups exhibited remarkable reductions in the latency period (53% & 44%) and grooming frequency . Moreover, marked elevations were observed in the ambulation frequency  and rearing frequency  in Safr+3-NP- and Cands+3-NP-treated groups, as compared to the 3-NP group, respectively.On the contrary, rats of Safr+3-NP and Cands+3-NP groups exhibited remarkable declines in the time spent in closed arms (13 and 11%) whereas marked elevations in the time spent in open arms (4- and 3-fold), the number of entries into open (1.8- and 1.7-fold) and closed arms (1.9- and 1.8-fold) as well as the total number of entries (1.8- and 1.7-fold), respectively, as compared to 3-NP group.The levels of 5-HT, 5-HIAA, NE and DA in striatum of all groups are recorded . Rats ofOn the other hand, the striatal levels of 5-HT, 5-HIAA, NE and DA in striatum of Safr+3-NP group were meaningfully higher than the 3-NP group, by 1.6 (P = 0.035), 3.8 (P = 0.000), 1.7 (P = 0.002) and 1.9-folds (P = 0.003), respectively. Similarly, Cands+3-NP group showed significant elevations in the striatal levels of 5-HT (P = 0.042), 5-HIAA (P = 0.000), NE (P = 0.001) and DA (P = 0.007), as compared to the 3-NP group, by 1.6, 4.0, 1.7 and 1.8-folds, respectively.The gene expression levels of MAO-A and MAO-B as well as the activities of MAO and AChE are reported .The striatal activity of AChE in 3-NP group showed a significant elevation by about 3-folds (P = 0.000), as compared to the CMC group. Nevertheless, rats of Safr+3-NP and Cands+3-NP groups showed marked declines in the activity of AChE by 29 (P = 0.003) and 22% (P = 0.000), respectively, as compared to the 3-NP group.In comparison to the CMC group, the rats of 3-NP group showed a remarkable elevation in the activity of MAO (2-folds) that was associated with marked upregulations in the expression levels of MAO-A (4.5-folds) and MAO-B (7.5–folds), in the striatum. In contrast, rats of Safr+3-NP and Cands+3-NP groups showed remarkable reductions in the activity of MAO (30 and 18%) and gene expression levels of MAO-A (63 and 58%) and MAO-B (71 and 64%), respectively, as compared to the 3-NP group.). In 3-NP group, significant elevations in the levels of NO  and MDA  were associated with a marked decline in the TAC , as compared to the corresponding control group.The striatal levels of NO, MDA and TAC in all groups are clarified . In 3-NPThough, in rats of Safr+3-NP and Cands+3-NP groups significant declines in the levels of NO (53 and 51%) and MDA (59 and 54%) were accompanied with a remarkable elevation in the TAC , respectively, as compared to 3-NP-induced rats.The activities of the mitochondrial complexes I, II and IV were displayed . In compOn the other hand, the levels of complexes I, II and IV in the striatum of Safr+3-NP and Cands+3-NP groups were remarkably (P = 0.000) greater than in 3-NP group (2.8 and 2.6-fold), (2.3 and 1.9-fold) along with (1.7 and 1.5-fold), respectively.In Comet assay parameters in striatal cells of all groups were analyzed . In 3-NPThe striatum area in the brain tissue of all the experimental groups were microscopically investigated Figs –9. In th, regarding the beneficial effects of Safr  on the movement defects induced by 3-NP (10 mg/kg), during the two weeks of experiment. However, our study was designed to investigate the possible mechanisms that might explain the potential protective roles of Safr and Cands against 3-NP-induced alterations. Accordingly, in the current study, the changes in behavioral, biochemical  as well as histopathological investigations were monitored.Our findings indicated that Safr (50mg/kg) and Cands (1mg/kg) may prevent or alleviate the progression HD and its associated impairments induced by the daily intraperitoneal injection of 3-NP (20 mg/kg), throughout the experimental duration (9 days). Our results are in accordance with the recent study by Fotoohi et al et al. . 3-NP injection can hinder the action of succinate dehydrogenase (SDH) in tricarboxylic acid cycle, leading to ATP depletion , leading to excessive metabolism of the stored glycogen and fats and subsequent weight loss . Moreover, 3-NP may reduce appetite through inducing striatal injuries .The reported weight reduction after 3-NP injection, in the present work, is in accordance with a previous study by Gao OFT, 3-NP altered ambulation, grooming and rearing frequencies, indicating impaired locomotor activity. Results of EPM and PA tests revealed that 3-NP also caused anxiety, altered motor activity as well as impaired memory. The decreased time spent in open arms, number of total and close arm entries by 3-NP rats may reflect the increased anxiety while reduced spontaneous motor activity . Several recent studies reported similar findings in 3-NP treated rats –44. In the brain, stress can boost angiotensin II (Ang II) levels , causing stimulation of Ang II receptor that increases Adrenocorticotropic Hormone (ACTH) secretion from the pituitary gland and subsequent release of corticosterone . In rodents, corticosterone can adversely affect cognitive memory . The observed behavioral disturbances were associated with remarkable declines in the striatal neurotransmitter levels in 3-NP treated rats. Mahdi et al also reported that 3-NP altered the levels of DA, 5-HT and NE in rats. These findings can be explained by the reported increase in the striatal MAO activity, in the 3-NP group, that is responsible for the breakdown of monoamines in the brain . MAO has two isoenzymes MAO-A (in catecholaminergic neurons) and MAO-B  . MAO catalyzes oxidative deamination of catecholamines (such as DA and NE) and 5-HT . The current results showed that 3-NP injection significantly upregulated the mRNA levels of MAO-A and -B.According to , as indicated in the preset study. Moslemi et al.  reported similar findings in striatum, cortex and hippocampus areas of male and female rats.AChE plays a key role in cholinergic transmission. Thus, the increased activity of AChE can reduce acetylcholine (ACh) level, leading to altered learning and memory activities . Mahdy et al.  also detected that 3-NP can induce oxidative damage in the striatum of rats, through enhancing ROS generation.Oxidative stress may play a major role in the 3-NP induced neurotoxicity, as manifested by recorded elevation in MDA level whereas a marked decline in TAC level, in striatum. MDA is a lipid peroxidation biomarker that is implicated in the cellular dysfunction in the brain under stress and neurodegenerative diseases , causing subsequent ROS excessive production. Moreover, 3-NP may inhibit the action of complexes I and IV by via S-nitrosation of their subunits .The current data showed significant suppression in the mitochondrial complexes I, II and IV, indicating 3-NP-induced mitochondrial dysfunction. 3-NP can irreversibly bind to complex II, leading to interruption of the respiratory chain , as ensured by the results of elevated plus maze. Renin–angiotensin system (RAS) is involved in the onset and progression of inflammation as well as oxidative damage in central nervous system (CNS), through boosting the Ang II level ,59. Noteworthy that the activation of RAS can upregulate inflammatory mediators that contribute to neurodegenerative disorders . In the same way, Ibrahim and Rasheed  recorded that 3-NP injection markedly upregulated iNOS and NO levels in the striatum of rats.3-NP injection caused a marked increase in NO level, indicating 3-NP induced neuroinflammation. This can be linked to the recorded increase in iNOS activity. NO can impair the spatial memory via promoting neuronal cell death . These findings were manifested by the reported DNA fragmentation. Similarly, Abdelfattah et al.  reported that 3-NP caused marked elevations in Casp-3 activity and Bcl-2-associated X protein (Bax) level, in the striatum. In addition, histopathological investigation showed nuclear pyknosis of neurons, indicating irreversible signs of apoptosis. Similar histopathological alterations were reported by Tasset et al. They attributed that to the 3-NP induced excessive generation of ROS.3-NP upregulated Casp-3 activity in striatum indicating 3-NP induced apoptosis. This could be linked to the increased level of FasL. FasL activates caspase cascade via binding to a tumor necrosis factor receptor (Fas). Casp-3 is a member of the caspase family that regulates the apoptotic process . The present data showed that Safr prevented the 3-NP-induced body weight loss. Similar results were obtained by Shahat et al. , in hyperthyroidism rat model after safr administration. They suggested that the sulfhydryl group in safr may interrupt thyroid hormone biosynthesis through decreasing iodide uptake, hydrogen peroxide level, and thyroid peroxidase activity. Noteworthy that the thyroid hormones are responsible for accelerating basal metabolic rate and protein wasting of the body.Safr is a monoterpene aldehyde that represents the major volatile component of safron. Safr gives saffron its characteristic aroma . This was ensured by the reported decline in the MDA level associated with a remarkable elevation in TAC levels. The free radical scavenging activity of safr can hinder the ROS-induced impairments in the neurotransmission . Moreover, safr can inhibit the reuptake of dopamine, serotonin and norepinephrine via direct binding to their receptors . In addition, safr caused significant reduction in the MAO activity, preventing the degradation of monoamines. The induced decline in MAO activity can be linked to the free radical scavenging action of safr. Noteworthy that MAO is hydrogen peroxide (H2O2)-dependent enzyme . Accordingly, the reported improvement in learning and memory activities, after safr treatment can be linked to the enhanced dopamine and serotonin levels as well as the improved blood flow to the brain . In the present study, Safr administration remarkably reduced anxiety as confirmed by the results of EPM test. This may be linked to the reported Safr-induced elevation in the striatal levels of monoamines including serotonin, that is responsible for the mood improvement . In the same line, Pontifex et al  reported that saffron extract had antidepressant and anti-anxiety properties. They attributed that to the improvement in anxiety-related behaviour were linked with the ability of saffron to affect the brain through inducing gut microbial shifts via gut–brain axis interactions. In addition, the spatial memory may be enhanced through Safr-enhanced AChE activity , as reported in the present study. In addition, the results of PA test revealed that spatial memory was improved by administration of Safr. This can be inked to the ability of Safr to reduce the apoptotic enzyme Casp-3 activity and MDA level, leading to reduced neuronal loss in the striatum. In the current study, Safr significantly improved locomotor activity as revealed by the results of OFT. These findings may be attributed to the ability of Safr to increase the striatal levels of norepinephrine and dopamine which are highly involved in motor control .Safr also prevented the 3-NP-induced reduction in the striatal levels of neurotransmitter. Safr has immense antioxidative properties through scavenging free radicals , as well as ischemia-reperfusion-enhanced damages in the cerebral cortex .As no histopathological damage was observed in the striatum of Safr-treated rats. Similarly, several previous studies illustrated the ameliorative role of Safr against oxidative damage-induced destructions in the hippocampus .In addition, Safr markedly reduced the Cas-3 and FasL levels, indicating its anti-apoptotic properties, in the current data. This can be linked to the antioxidative properties of Safr. In addition, Safr can inhibit the release of cytochrome c, leading to inactivation of Cas-3  reported that Safr treatment caused remarkable decline in iNOS and peroxynitrite levels, besides restoration of mitochondrial function and DNA integrity. Koul and Abraham  reported that Safr protected the bone marrow of mice against the DNA damaging effect of gamma radiation. They attributed that to its antigenotoxic properties. Safr may protect DNA from harmful damage through external binding or intercalative styles .The iNOS level significantly declined in the Safr-treated rats before 3-NP. This may explain the reduced levels of NO, reflecting its anti-inflammatory action. In the same manner, Bukhari et al. et al.  also reported that an oral dose of 5 mg/kg/day prevented body weight loss in rats under chronic stress. Furthermore, Cands treatment improved motor activity and exploratory behavior as indicated by the results of OFT. This can be also linked to the reported elevation in NE and DA levels in striatum after treatment with Cands. In addition, Cands enhanced spontaneous motor activity and reduced 3-NP-fear drive, as indicated by EPM results, indicating anti-anxiety action of Cands. This can be due to the ability of increase the striatal serotonin levels, in the present data. PA results revealed that Cands improved the spatial memory. Braszko et al.  also recorded that Cands prevented the stress-induced memory impairment in rats. In the same manner, Thakur et al.  recorded that administration of Cands at doses of 3 and 5 mg/kg remarkably improved the observed changes induced by haloperidol in rearing and grooming behavior as well as retention time in the open arms of EPM. The improved performance of rats in the behavioral tests after treatment with Cands may be linked to enhanced cholinergic system as confirmed by the reported reduction in AChE activity and subsequent increase in acetylcholine. Previous studies showed that Ang II receptor blockers can improve cognitive performance via preventing Ang II inhibitory action on the release of acetylcholine from entorhinal cortex .The current results showed that Cands hindered 3-NP-induced body weight loss. Chen . Thus, Cands can reduce H2O2 generation leading to decreasing the 3-NP-induced oxidative stress. This may explain reported reduction in MDA levels whereas increased TAC levels in striatum, after treatment with Cands. Similar findings were reported by Tota et al, in the brain of mice after an intraperitoneal injection with 0.1 mg/kg in mice. They linked that to the ability of Cands to scavenge free radicals. Cands has the ability to inhibit the ROS production by increasing the glutathione (GSH) content and normalizing the redox status . Bețiu et al stated that Cands at doses of 0.1–0.3mg/kg for 7 days remarkably alleviated mitochondrial electron transport system dysfunction. This was ensured by the improved Complexes I, II and IV, after treatment with Cands, in the present study. Moreover, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase induces oxidative damage through enhancing superoxide anion generation , after being activated by binding of Ang II to AT1 receptors . Thus, Cands can also indirectly hinder 3-NP-induced oxidative damage via blocking Ang II receptors .One of the proposed mechanisms of action of Cands can be through preventing oxidative damage. Cands administration remarkably reduced the 3-NP induced elevation in the MAO activity. This could be linked to the reported Cands-induced downregulation of MAO. As MAO is responsible for H2O2 generation et al.  described that an oral dose of 1 mg/kg of candesartan markedly reduced LPS-induced inflammation in the brain. Thakur et al.  detected that Cands has anti-inflammatory actions against haloperidol-induced inflammation in rats. They linked that to its ability to downregulate the expression of proinflammatory cytokines such as IL-1β and pro-inflammatory mediators as tumor necrosis factor-alfa (TNF-α).Cands treatment also prevented the 3-NP-induced elevations in striatal level of NO, reflecting its anti-inflammatory action. This finding can be ascribed to the reported reduction in iNOS activity after treatment with cands. Likewise, Benicky et al.  reported that cands as other ARB have antioxidative and anti-apoptotic effects. ARBs such as Cands have been reported to show their neuroprotective activity through regulating key proteins such as neurotrophic factors and dopamine transporter . These proteins are highly involved in the progressive degeneration of dopaminergic system as reported in many neurodegenerative diseases . Accordingly, cands can penetrate blood brain barrier (BBB) and modulate 3-NP-induced HD symptoms via inhibiting the stimulating action of Ang II on dopamine receptors, preventing neuronal cell death .Cands also caused marked decline in the Cas-3 activity and FasL level, indicating anti-apoptotic properties. Thakur Collectively, the current study demonstrated that the 3-NP-induced changes in the levels of neurotransmitters, oxidative stress indices and inflammatory mediators led to a decrease in the cognitive and locomotor activities. Interestingly, co-administration of Safr or Cands along with 3-NP was found to minimize or even prevent the biochemical, behavioral and histological disturbances in the experimented rats. Safr was mostly more effective than Cands regarding behavioral tests, MAO, AChE, TAC and DNA damage. Consequently, this research suggests that Safr and Cands exert neuroprotective effects in a rat model of 3-NP-induced HD and hence might have potential as drug candidates for the treatment of HD. 20 Apr 2023 <div> PONE-D-23-06048 </div>  <div> Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington’s disease </div>  <div> PLOS ONEDear Dr. Risk,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. 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Reviewer #1: The manuscript “Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington's disease” clarifies the role of Safranal and Candesartan in prevention of the progression of Huntington's disease and its associated complications through their antioxidant, anti-inflammatory, anti-apoptotic and neuromodulator effects. I think the manuscript is of good idea. However, the manuscript needs some changes to be taken into consideration.• The authors should clarify in the abstract the timing and duration of drugs administration as well as 3-NP. The authors also could add a figure for the experimental design in the methodology.• Kindly modify the Guide for the Care and Use of Laboratory Animals 129 published by the US National Institute of Health  to a more recent one .• The authors should explain the reason for using these large number of animals (40 rats per group)• The authors should provide references for estimation of complexes I and II• The authors could add a recent reference demonstrating the role of safranal in neurodegenerative disease (PMID: 33611726).• Kindly check that all abbreviations are defined before being used like RIPA, TBS, ……etc• The manuscript needs to be edited for few awkward sentences and grammar mistakesReviewer #2: REVIEW REPORTManuscript number: PONE-D-23-06048Full Title: Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington’s diseaseAuthors assessed the effect of safranal and candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington’s disease. This is an interesting subject area. Based on the findings of the research, authors concluded that safranal and candesartan may prevent or delay the progression of HD and its associated impairments through their antioxidant, anti-inflammatory, anti-apoptotic and neuromodulator effects. Although the manuscript has been well written, some clarifications or inputs need to be made in order to greatly improve the quality of this manuscript. Below are some of the comments.Abstract1. A statement on the potential neuroprotective effects of safranal and candesartan should be inserted before the aim to justify why these drugs were chosen.2. The doses of the test compounds should be well written. Are authors referring to 50 mg/kg for safranal and 1 mg/kg for candesartan? In line 48, authors should indicate the route of administration for 3-NP.3. The sentence in Lines 48-50 is quite confusing. Description of methods should always be precise and easy to apprehend. Authors should kindly revise.Introduction4. In line 65, CAG should be defined. It is always important to define abbreviations in a text on first mention. The sentence captured in lines 88-90 is incomplete. Please revise.Materials and Methods5. In addition to weight, can authors state the age of animals. Were the animals housed individually or in groups during the acclimatization period? If in groups, how many were housed in a group? Also provide cage dimensions. Authors should indicate the start times of the light and dark cycle.6. Number of animals (40) in a group are too many, bringing about issues regarding ethics. Why 40? How were drug solutions prepared and in what volume were they administered? What was the rationale of administering paraffin oil and CMC to some groups of rats? Why wasn’t a naïve control group introduced. How were drugs administered orally? What informed the choice of doses for safranal and candesartan? In addition, why did authors decide to use single doses for the test compounds? Why did authors choose to administer test compounds for 9 days?7. Why did group 5 receive 3-NP from day 3 and not day 1? The statement “The sixth group (Safr+3-NP) received IP injection of 50 mg Safr/kg/day for 9 days followed by IP injection of 20 mg 3-NP/kg/day from the 3rd day to the 9th day’ is difficult to appreciate. Same can be said for group 7. Authors should clarify exactly how the dosing of 3-NP plus test compounds was done.8. With regards to the behavioural tests performed, can authors indicate the exact start day and time of the experiments. For example, EPM was conducted on day 10 or 11 and at 9 a.m. after start of treatment etc.9. OFT. Why was wood and not Plexiglas used for the OF apparatus? Authors indicate that the OFT was conducted in morning daylight. What was the exact time? Dimension for the OF apparatus: (90 x 90 x 25 cm3) or (90 cm x 90 cm x 90 cm) not (90 x 90 x 25 cm). same applies to the squares. What action was taken after the 5-minute session if an animal defecated in the arena? How were the parameters measured? The OFT is a conventional approach/avoidance paradigm in which anxiety and exploration are simultaneously evoked by a novel setting. An increase in activity or time spent in the center of the open field indicates reduction in anxiety and/or increase in exploration. It would have therefore been very important for authors to measure central activity.10. EPM. The description indicates that anxiety behaviour was measured, not cognitive function. To assess cognitive function, the EPM transfer latency can be used. Please refer to literature. Was the apparatus made of wood or Plexiglas? How were the parameters recorded? With regards to dimensions, refer to comments on OF.11. Experiments to measure movement behaviour should have been performed to assess the effects of safranal and Candesartan on motor abnormalities since this is an important trait of HD. Refer to Sidhu et al, 2018.12. With regards to the one-way ANOVA, which post hoc test was used for multiple comparisons.Results13. What were the observed effects after 3-NP administration?14. In line 277, which corresponding control group are authors referring to?15. Statistical analysis e.g. p values are missing in the description.16. Too many tables. Presentation of results as a blend of figures and tables would have been the best. Figures most often are easy to interpret. Ill suggest that some results from the behavioural tests are presented as figures.17. For all tables, the statement “Data is displayed as mean ± standard error (SE)” can be captured in the legend.18. For all tables, authors should note that the significant differences that exist with regards to the various comparisons featured in the legend have not been duly captured. For instance, e: represents significant difference as compared to 3-NP-treated group. Looking, at the values for PO or CMC in table 2, there should be significance in some of the parameters.19. With regards to figs 1-4 (Photomicrograph of the striatum for the various treatments), authors can show in the micrographs, the normal and histological changes of neurons in the striatum for better appreciation.Discussion20. Authors made reference to a study by Fotoohi et al, regarding the beneficial effects of Safranal on movement defects induced by 3-NP during the two weeks of experiment. The doses used in this study are quite higher that that study ? Why didn’t authors use the same or similar doses since beneficial effects were achieved with them.21. There is silence on the relevance of assessing the effects of the test compounds in the EPM and OFT. A detailed discussion is needed. As stated earlier, EPM test measures anxiety behaviour but can also be used to measure cognitive function in the transfer latency test. This wasn’t done. It is therefore surprising authors are relating EPM to cognitive impairment.22. In paragraph 2, authors conclude by stating “the current results showed that 3-NP injection significantly upregulated the mRNA levels of MAO-A and –B.” What about the effects of test compounds? Authors should focus their discussion on the benefits of test drugs based on the results.********** <font color="black"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1: NoReviewer #2: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, AttachmentPONE-D-23-06048.docxSubmitted filename: Click here for additional data file. 16 Jul 2023Ref.: Ms. No. PONE-D-23-06048Title: “Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington’s disease”.Journal: PLOS ONEDear editor-in chief, Respected Editor of The Journal of PLOS ONEWe appreciate the valuable inputs given by the respected reviewers which helped us greatly to improve the manuscript. We have tackled all the recommended changes. Below, you will find answers item-by-item to the reviewers’ comments:________________________________________Dear respected reviewers, On behalf of my coauthors, I am so grateful to you for your thorough revision and helpful insights that will certainly support our manuscript. Reviewer #1:Comment #1: The authors should clarify in the abstract the timing and duration of drugs administration as well as 3-NP. The authors also could add a figure for the experimental design in the methodology.Response: In the revised manuscript, under Abstract section, page 2; lines 49-53, all the timings and durations of drug and 3-NP administrations were illustrated.In the revised manuscript, page 5, line 132, fig 1 was added as a diagrammatic representation of the experimental design.Comment #2: Kindly modify the Guide for the Care and Use of Laboratory Animals 129 published by the US National Institute of Health  to a more recent one .Response: In the revised manuscript, under M&M, page 5, line 138, the necessary changes were considered and the  was replaced by the more recent one .Comment #3: The authors should explain the reason for using these large number of animals (40 rats per group).Response: All the studied parameters were measured in the striatum that is a very small part of the brain weighing (50-100 mg). In order to measure each of the studied parameters striatum should be homogenized in a special buffer. For example, oxidative parameters (1.15% KCl), MAO (0.2M Phosphate buffer), ACHE (20mM Phosphate buffer), monoamines (n-acidified butanol),…etc, Accordingly, there was a need to take a large sample size to cover all the required parameters.Comment #4: The authors should provide references for estimation of complexes I and II.Response: In the revised manuscript, under M&M section, page 8, lines 217 and 230, the following references for the techniques of complexes I and II were cited.Horowitz JD, Chong CR, Ngo DT, Sverdlov AL. Effects of acute hyperglycaemia on cardiovascular homeostasis: does a spoonful of sugar make the flow-mediated dilatation go down? J Thorac Dis 7: E607–E611, 2015. doi: 10.3978/j.issn.2072-1439.2015.12.40.Ansari F, Yoval-Sánchez B, Niatsetskaya Z, Sosunov S, Stepanova A, Garcia C, Owusu-Ansah E, Ten V, Wittig I, Galkin A. Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples. J Biol Chem. 2021 Oct;297(4):101204. doi: 10.1016/j.jbc.2021.101204.Comment #5: The authors could add a recent reference demonstrating the role of safranal in neurodegenerative disease (PMID: 33611726).Response: In the revised manuscript, under Discussion section, line 479, page 23, the suggested reference (PMID: 33611726) is utilized to illustrate the role of safranal in neurodegenerative disease in comparison with the present study.Comment #6: Kindly check that all abbreviations are defined before being used like RIPA, TBS, ……etc.Response: in the revised manuscript, all the undefined abbreviations were defined and highlighted under M&M section, page 7 , page 8 (line 204-209) and page 9 (line 247 & 258), as well as under discussion section, page 23 (line 494), page 24 (lines 518 & 526) and page 26 (lines 578 & 586), as recommended.Comment #7: The manuscript needs to be edited for few awkward sentences and grammar mistakes.Response: All the manuscript items were carefully and thoroughly revised for language and grammatical mistakes, as per comment. Reviewer #2: AbstractComment #1: A statement on the potential neuroprotective effects of safranal and candesartan should be inserted before the aim to justify why these drugs were chosen.Response: in the revised manuscript, page 2, lines 44-46, a statement was added as per request. "Safranal (Safr) that is found in saffron essential oil has antioxidant, anti-inflammatory and anti-apoptotic actions. Candesartan (Cands) is an angiotensin receptor blocker that has the potential to prevent cognitive deficits." Comment #2: The doses of the test compounds should be well written. Are authors referring to 50 mg/kg for safranal and 1 mg/kg for candesartan? In line 48, authors should indicate the route of administration for 3-NP.Response: in the revised manuscript, page 2, lines51-53, the applied doses and routes of administration were clarified and highlighted.Comment #3: The sentence in Lines 48-50 is quite confusing. Description of methods should always be precise and easy to apprehend. Authors should kindly revise.Response: in the revised manuscript, page 2, lines 51-53, description of the experimental groups was revised as per comment.IntroductionComment #4: In line 65, CAG should be defined. It is always important to define abbreviations in a text on first mention. The sentence captured in lines 88-90 is incomplete. Please revise.Response: in the revised manuscript, page 2, line 69, CAG was defined as cytosine–adenine–guanine. In addition, in the revised manuscript, page 3, lines 91-93, the sentence was revised and completed.Materials and MethodsComment #5: In addition to weight, can authors state the age of animals. Were the animals housed individually or in groups during the acclimatization period? If in groups, how many were housed in a group? Also provide cage dimensions. Authors should indicate the start times of the light and dark cycle.Response: in the revised manuscript, page 4, line 113, the age of rats was stated. In page 4, lines 114-115, the number of animals per cage as well as the dimensions of the cage were stated. In page 4, lines 116-117, the starting time of light and dark cycle were stated.Comment #6: Number of animals (40) in a group are too many, bringing about issues regarding ethics. Why 40? How were drug solutions prepared and in what volume were they administered? What was the rationale of administering paraffin oil and CMC to some groups of rats? Why wasn’t a naïve control group introduced. How were drugs administered orally? What informed the choice of doses for safranal and candesartan? In addition, why did authors decide to use single doses for the test compounds? Why did authors choose to administer test compounds for 9 days?Response: 1) Regarding te sample size, all the studied parameters were measured in the striatum that is a very small part of the brain weighing (50-100 mg). In order to measure each of the studied parameters striatum should be homogenized in a special buffer. For example, oxidative parameters (1.15% KCl), MAO (0.2M Phosphate buffer), ACHE (20mM Phosphate buffer), monoamines (n-acidified butanol),…etc, Accordingly, there was a need to take a large sample size to cover all the required parameters.2) Regarding dose preparation of drugs:a) Preparation of 3-NP (20 mg/kg)Each 20mg of 3-NP was dissolved in 2.0 mL of 0.9% saline. The volume of administration was 2.0 mL/kg body weight.b) Preparation of Safr (50mg/kg)Each 50mg of Safr was dissolved in 2.0 mL of paraffin oil. The volume of administration was 2.0 mL/kg body weight.c) Preparation of Cands (1mg/kg)Each 1mg of Cands was dissolved in 2.0 mL of CMC. The volume of administration was 2.0 mL/kg body weight.3) Regarding the used vehicles, note that CMC (carboxymethyl cellulose) is an inert vehicle that is suitable for giving homogenous suspension of Candsartan. CMC is safe for most animal species . However, safranal is an oil that is miscible in paraffin oil (an inert vehicle). Parafin oil was safe to rodents . Thus, there was no need to use naïve control group. 4) Regarding the administration of signle doses, each test chemical was administered daily with the selected dose (a single dose per day).5) In our study, the applied dose of 3-NP , safranal  and Candsartan (1mg/kg) were selected according to Sharma et al. (2012), Shahat et al. (2022), Ishrat et al., 2022, respectively. According to Sharma et al. (2018), an intraperitoneal injection of 20 mg of 3-NP/kg/day, for 7 days, is adequate to induce the HD model. Shahat et al. (2022) concluded that a daily injection of 50 mg safranal/kg, for 3 weeks, prevented the L-thyroxine-induced deleterious effects on the brain and improved its antioxidant status. According to Ishrat et al (2022), Cands could protect the rat's brain against stroke after 72h of administration.6) In addition, Mehdizadeh et al (2013) reported that safranal extract at doses of 20 mg -160 mg/kg, for 9 days, exerted protective action against Isoproterenol-Induced Myocardial Infarction in Wistar Rats.Shahat AS, Hassan WA, El-Sayed WM. N-Acetylcysteine and Safranal prevented the brain damage induced by hyperthyroidism in adult male rats. Nutr Neurosci. 2022;25(2):231-245.https://doi.org/10.2903/j.efsa.2020.6211EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Bampidis, V., Azimonti, G., Bastos, M. L., Christensen, H., Dusemund, B., Kos Durjava, M., Kouba, M., López-Alonso, M., López Puente, S., Marcon, F., Mayo, B., Pechová, A., Petkova, M., Ramos, F., Sanz, Y., Villa, R. E., Woutersen, R., Bories, G., Gropp, J., … Aquilina, G. (2020). Safety and efficacy of sodium carboxymethyl cellulose for all animal species. EFSA journal. European Food Safety Authority, 18(7), e06211. Mehdizadeh, R., Parizadeh, M. R., Khooei, A. R., Mehri, S., & Hosseinzadeh, H. (2013). Cardioprotective effect of saffron extract and safranal in isoproterenol-induced myocardial infarction in wistar rats. Iranian journal of basic medical sciences, 16(1), 56.Sharma, M., Sharma, N., & Sharma, R. (2012). Neuroprotective effect of Zingiber officinale in 3-np-induced huntington disease. IOSR Journal of Pharmacy, 2(6), 61-70.Ishrat T, Soliman S, Eldahshan W, Pillai B, Ergul A, Fagan SC. Silencing VEGF-B diminishes the neuroprotective effect of candesartan treatment after experimental focal cerebral ischemia. Neurochem Res. 2018;43, 1869–1878. Comment #7: Why did group 5 receive 3-NP from day 3 and not day 1? The statement “The sixth group (Safr+3-NP) received IP injection of 50 mg Safr/kg/day for 9 days followed by IP injection of 20 mg 3-NP/kg/day from the 3rd day to the 9th day’ is difficult to appreciate. Same can be said for group 7. Authors should clarify exactly how the dosing of 3-NP plus test compounds was done.Response: The study aims to investigate the potential protective roles of Safr and Cands. Accordingly, the authors tend to start the treatments before the induction of HD model with two days. On the third day, a dose of 20 mg/kg for 7 days is enough to induce HD model in Wister rats, according to Sharma et al. (2012).In the revised manuscript, page 4, lines 128 -131, the description of groups 6 and 7 was revised as per comment.Sharma, M., Sharma, N., & Sharma, R. (2012). Neuroprotective effect of Zingiber officinale in 3-np-induced huntington disease. IOSR Journal of Pharmacy, 2(6), 61-70.Comment #8: With regards to the behavioural tests performed, can authors indicate the exact start day and time of the experiments. For example, EPM was conducted on day 10 or 11 and at 9 a.m. after start of treatment etc.Response: in the revised manuscript, page 5-6, lines 145, 155, 161 and 164, the starting time of each experiment was stated and highlighted.Comment #9: OFT. Why was wood and not Plexiglas used for the OF apparatus? Authors indicate that the OFT was conducted in morning daylight. What was the exact time? Dimension for the OF apparatus: (90 x 90 x 25 cm3) or (90 cm x 90 cm x 90 cm) not (90 x 90 x 25 cm). same applies to the squares. What action was taken after the 5-minute session if an animal defecated in the arena? How were the parameters measured? The OFT is a conventional approach/avoidance paradigm in which anxiety and exploration are simultaneously evoked by a novel setting. An increase in activity or time spent in the center of the open field indicates reduction in anxiety and/or increase in exploration. It would have therefore been very important for authors to measure central activity.Response: in the revised manuscript, page 5, line 143, the dimensions of OF apparatus were corrected. In page 5, lines 147-150, the action after each session was stated. Regarding the central activity, that is very interesting and should be taken into consideration but here in our study we focused on using OFT to measure the effects on general motor activity.According to Okaichi et al., (2006), wooden OF apparatus is suitable for measuring ambulation frequency. Lamprea et al. (2008) also used wooden OF to measure locomotor activity.Okaichi, Y., Amano, S., Ihara, N., Hayase, Y., Tazumi, T., & Okaichi, H. (2006). Open‐field behaviors and water‐maze learning in the F substrain of Ihara epileptic rats. Epilepsia, 47(1), 55-63.Lamprea, M. R., Cardenas, F. P., Setem, J., & Morato, S. (2008). Thigmotactic responses in an open-field. Brazilian Journal of Medical and Biological Research, 41, 135-140.Comment #10: EPM. The description indicates that anxiety behaviour was measured, not cognitive function. To assess cognitive function, the EPM transfer latency can be used. Please refer to literature. Was the apparatus made of wood or Plexiglas? How were the parameters recorded? With regards to dimensions, refer to comments on OF.Response: in the revised manuscript, page 6, line 152, the aim of the EPM test was changed as to copy with measured parameters of EPM. As open arm time and number of entries are measures of anxiety while total or/and close arm entries represent measures of the spontaneous motor activity .In the revised manuscript, page 5, lines 152-156, the apparatus material, dimensions and how parameters measured are all listed and highlighted as per comment. https://doi.org/10.1038/nprot.2007.44Walf, A. A., & Frye, C. A. (2007). The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nature protocols, 2(2), 322–328. Comment #11: Experiments to measure movement behaviour should have been performed to assess the effects of safranal and Candesartan on motor abnormalities since this is an important trait of HD. Refer to Sidhu et al, 2018.Response: of course, measuring the movement behaviour is so important to assess the effectiveness of the used drugs against the HD model. Thus, in the present study, we utilized open field test, as a test for general locomotion .https://doi.org/10.3791/55491Osmon, K. J., Vyas, M., Woodley, E., Thompson, P., & Walia, J. S. (2018). Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice. Journal of visualized experiments : JoVE, (131), 55491. Comment #12: With regards to the one-way ANOVA, which post hoc test was used for multiple comparisons.Response: in the revised manuscript, page 9, line 256, the applied post hoc test was illustrated as duncan's test.ResultsComment #13: What were the observed effects after 3-NP administration?Response: in the revised manuscript, RESULT section, pages 9, 12, 16 and 17, each paragraph started with describing the effect of 3-NP on the studied parameters. Comment #14: In line 277, which corresponding control group are authors referring to?Response: in the revised manuscript, page 11, line 291, the corresponding control group here refers to the second group (control for Cands and 3-NP), as clarified in experimental design, page 4-5. Comment #15: Statistical analysis e.g. p values are missing in the description.Response: in the revised manuscript, result section, action was taken, p-values were added to the comments.Comment #16: Too many tables. Presentation of results as a blend of figures and tables would have been the best. Figures most often are easy to interpret. Ill suggest that some results from the behavioural tests are presented as figures.Response: in the revised manuscript, pages 10 – 13, the tables of behavioral tests were replaced by figures 2-5.Comment #17: For all tables, the statement “Data is displayed as mean ± standard error (SE)” can be captured in the legend.Response: in the revised manuscript, action was taken in all tables.Comment #18: For all tables, authors should note that the significant differences that exist with regards to the various comparisons featured in the legend have not been duly captured. For instance, e: represents significant difference as compared to 3-NP-treated group. Looking, at the values for PO or CMC in table 2, there should be significance in some of the parameters.Response: statistical test was changed to Duncan's test for better clarification.Comment #19: With regards to figs 1-4 (Photomicrograph of the striatum for the various treatments), authors can show in the micrographs, the normal and histological changes of neurons in the striatum for better appreciation.Response: In the figures file, the arrows were added to the figures to illustrate the normal structure of the striatum as well as the histopathological changes in the experimental groups.DiscussionComment #20: Authors made reference to a study by Fotoohi et al, regarding the beneficial effects of Safranal on movement defects induced by 3-NP during the two weeks of experiment. The doses used in this study are quite higher that that study ? Why didn’t authors use the same or similar doses since beneficial effects were achieved with them.Response: In our study, the applied dose of 3-NP  and safranal  and were selected according to Sharma et al. (2012) and Shahat et al. (2022), respectively. According to Sidhu et al. (2018), an intraperitoneal injection of 20 mg of 3-NP/kg/day, for 7 days, is adequate to induce the HD model. Shahat et al. (2022) concluded that a daily injection of 50 mg safranal/kg, for 3 weeks, prevented the L-thyroxine-induced deleterious effects on the brain and improved its antioxidant status.Shahat AS, Hassan WA, El-Sayed WM. N-Acetylcysteine and Safranal prevented the brain damage induced by hyperthyroidism in adult male rats. Nutr Neurosci. 2022;25(2):231-245.Sharma, M., Sharma, N., & Sharma, R. (2012). Neuroprotective effect of Zingiber officinale in 3-np-induced huntington disease. IOSR Journal of Pharmacy, 2(6), 61-70.Comment #21: There is silence on the relevance of assessing the effects of the test compounds in the EPM and OFT. A detailed discussion is needed. As stated earlier, EPM test measures anxiety behaviour but can also be used to measure cognitive function in the transfer latency test. This wasn’t done. It is therefore surprising authors are relating EPM to cognitive impairment.Response: in the revised manuscript, page 22, lines 497-499, the estimated output of EPM was changed as per advice.Comment #22: In paragraph 2, authors conclude by stating “the current results showed that 3-NP injection significantly upregulated the mRNA levels of MAO-A and –B.” What about the effects of test compounds? Authors should focus their discussion on the benefits of test drugs based on the results.Response: in the revised manuscript, page 25, lines 540-543 as well as page 26, lines 572-574, the discussion of safr and Cands was revised and highlighted.________________________________________We hope that we have answered all the comments raised by the respected referees and followed their recommendations and adopted the manuscript on journal's guidelines/style. We think that our manuscript has been considerably improved because of these thoroughly revisions. We hope that our revised manuscript “Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington’s disease” is now suitable for publication in your respected journal “PLOS ONE”.We should like to take this opportunity to thank the editor for his dedication and support. We look forward to hearing from you regarding our submission.Thank you for understanding and supportRespectfullySherine Maher Rizk, PhD Biochemistry DepartmentFaculty of PharmacyCairo University nagwa.mahmoud@pharma.cu.edu.egE-mail: AttachmentResponse to reviewers 2.docxSubmitted filename: Click here for additional data file. 23 Aug 2023 <div> PONE-D-23-06048R1 </div>  <div> Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington’s disease </div>  <div> PLOS ONEDear Dr. Risk,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. 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Details regarding preparation of the various drug solutions should be outlined in the manuscript (This is captured in your response)3. For the experimental design/procedure, authors can represent the various treatments as Safr 50 mg/kg/day rather than 50 mg Safr/kg/day.4. For the various dimensions (cm3), 3 should be in superscript.5. With regards to the behavioural testing, authors indicate that the training session for the passive avoidance test started at 6:00 pm on training day 9 in which a 0.5 mA with 50 Hz foot electric shock was delivered for two seconds. The behavioural assessment in the EPM & OFT was however done on day 10 of treatment. Couldn’t this have influenced the results seen with regards to the EPM and OFT? In line 149, can authors state the % alcohol used?6. In Fig 1, the experimental design should capture a detailed sequential summary of all experiments from start to finish. This includes treatments, behavioural assessments & sample analysis.Results7. What were the observed effects after 3-NP administration?8. Capital letters are used to represent the outcomes of the statistical analysis. Can authors use symbols instead? Maybe *, #, etc.9. Symbol for seconds is “s”.10. The y-axis of the various figures can be numbered without decimals.11. Authors used different in-text citations for the figures. Eg. fig 2, fig(5), or Fig. 5. Authors should refer to the journal’s guidelines and be consistent.Discussion12. There is silence on the relevance of assessing the effects of the test compounds in the EPM and OFT.**********  <font color="black"> 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? 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Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  16 Oct 2023Ref.: Ms. No. PONE-D-23-06048R1Title: “Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington’s disease”.Journal: PLOS ONEDear editor-in chief, Respected Editor of The Journal of PLOS ONEWe appreciate the valuable inputs given by the respected reviewers which helped us greatly to improve the manuscript. We have tackled all the recommended changes. Below, you will find answers item-by-item to the reviewers’ comments:________________________________________Dear respected reviewers, On behalf of my coauthors, I am so grateful to you for your thorough revision and helpful insights that will certainly support our manuscript. Reviewer #2:IntroductionComment #1: The sentence captured in lines 91-93 ‘’Ang II type 1 receptor (AT1R) blockers (ARBs) have been reported to be beneficial in attenuating cognitive deficits observed in Alzheimer’s disease, Parkinson’s disease, vascular’’ is incomplete. Please revise.Response: In the revised manuscript, Introduction section, page 3, lines 92-94, the captured sentence was revised as follows:"Ang II type 1 receptor (AT1R) blockers (ARBs) have been reported to be useful in diminishing cognitive deficits linked to Post-Stroke Cognitive Impairment (PSCI), Alzheimer’s Disease, Parkinson’s Disease, and Vascular Cognitive Impairment (VCI)"Materials and MethodsComment #2: Details regarding preparation of the various drug solutions should be outlined in the manuscript (This is captured in your response).Response: In the revised manuscript, M&M section, page 4, lines 114-121, the details of drug preparations were stated for each of the applied drugs.Comment #3: For the experimental design/procedure, authors can represent the various treatments as Safr 50 mg/kg/day rather than 50 mg Safr/kg/day.Response: In the revised manuscript, M&M section, page 5, lines 134-140, the rcommended comments in the experimental design/procedure were considered.Comment #4: For the various dimensions (cm3), 3 should be in superscript.Response: In the revised manuscript, M&M section, page 4 (line 125), page 6 (lines 154- 171), action was taken as per comment.Comment #5: With regards to the behavioural testing, authors indicate that the training session for the passive avoidance test started at 6:00 pm on training day 9 in which a 0.5 mA with 50 Hz foot electric shock was delivered for two seconds. The behavioural assessment in the EPM & OFT was however done on day 10 of treatment. Couldn’t this have influenced the results seen with regards to the EPM and OFT? In line 149, can authors state the % alcohol used?Response: in the revised manuscript, M&M section (fig 1), pages 5, line 139, in the summary of the experimental design, a flowchart was added to show that each of the behavioural tests  was conducted using different set of the experimental animals . Actually, we had two options; option #1 is to conduct the OFT and EPM on the 10th day, then to run the training session of PA, on the 11th day, then the main experiment of PA on the 12th day. Accordingly, three days would be passed after the last treatment. thus, the results of PA may not be representative for the conducted treatment. Option #2 is to conduct the behavioural experiments using different set of the experimental animals , to avoid the effect of the electrical shock in the PA on the results of the other behavioral tests. We chosen the second option, because it gave us the opportunity to apply all the beahvioural tests on the 10th day and to avoid that any of the studied behaviors may be affected by the other behavioural tests.In the revised manuscript, M&M section, page 6, line 161, the % of alcohol was stated as 70%.Comment #6: In Fig 1, the experimental design should capture a detailed sequential summary of all experiments from start to finish. This includes treatments, behavioural assessments & sample analysis.Response: in the revised manuscript, M&M section, page 5, lines 139-140, the detailed sequential summary of all the experiments was stated as per comment.ResultsComment #7: What were the observed effects after 3-NP administration?Response: in the revised manuscript, Results section, page 10, lines 277-280, a brief description of the the effect of 3-NP injection was written. In addition, in the subsequent paragarphs, the detailed description of the effect of 3-NP inection was written and highlighted in yellow.Comment #8: Capital letters are used to represent the outcomes of the statistical analysis. Can authors use symbols instead? Maybe *, #, etc.Response: As clarified in page 10, line 267, under M&M-statistical analysis, the applied post-hoc test was Duncan's test to study the statistical homogeneity among all the experimental groups. Accordingly, letters are best fitting to represent the outcomes. However, to use symbols like "*, #,…", are best fitting for the outcome of other statistical post-hoc tests like for example tukey HSD and LSD. Duncan's test gives us the opportunity to compare among all groups, using Alphabet letters (in ascending or desecending order), especially that the groups have multiple controls. However, the outcomes of LSD and HSD may cause some confusion because there will be at least 6 symbols  Comment #9: Symbol for seconds is “s”.Response: In the revised manuscript, M&M section, page 6-7 , as well as in Results section, page 12 , page 14  and page 15 , action was taken as per comment.Comment #10: The y-axis of the various figures can be numbered without decimals.Response: In the revised manuscript, Results section, page 11 , page 12 , page 14  and page 15 , all decimals were removed as per comment.Comment #11: Authors used different in-text citations for the figures. Eg. fig 2, fig(5), or Fig. 5. Authors should refer to the journal’s guidelines and be consistent.Response: In the revised manuscript, pages 4-5 , under M&M section as well as in page 10-15 , under the results section, all Figs were adjusted according to the journal guidelines.DiscussionComment #12: There is silence on the relevance of assessing the effects of the test compounds in the EPM and OFT.Response: In the revised manuscript, in Discussion section, page 26 (lines 565-576) and page 27 (lines 590-602), action was taken as per comment.________________________________________We hope that we have answered all the comments raised by the respected referees and followed their recommendations and adopted the manuscript on journal's guidelines/style. We think that our manuscript has been considerably improved because of these thoroughly revisions. We hope that our revised manuscript “Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington’s disease” is now suitable for publication in your respected journal “PLOS ONE”.We should like to take this opportunity to thank the editor for his dedication and support. We look forward to hearing from you regarding our submission.Thank you for understanding and supportRespectfullySherine Maher Rizk, PhD Biochemistry DepartmentFaculty of PharmacyCairo University sherine.abdelaziz@cu.edu.egE-mail: AttachmentResponse to reviewers R2 222.docxSubmitted filename: Click here for additional data file. 18 Oct 2023Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington’s diseasePONE-D-23-06048R2Dear Dr. Risk,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Yasmina Abd‐ElhakimAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 24 Oct 2023PONE-D-23-06048R2 Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington’s disease Dear Dr. Rizk:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProf. Dr. Yasmina Abd‐Elhakim Academic EditorPLOS ONE